1. Field of the Invention
The present invention relates to an apparatus for performing a continuous free flow electrophoresis process. More particularly, the present invention relates to an apparatus for performing a continuous free flow electrophoresis procedure in which means are provided for controlling the buffer fluid flow across the separation chamber.
2. Description of the Prior Art
Electrophoresis in general is the phenomenon of the migration of charged particles or ions in a liquid carrier (buffer) medium under the influence of an electrical field. This phenomenon can be used to separate the various components of a biological sample comprised of various small particles which, by reason of different surface chemical properties, exhibit different concentrations of surface charge in the given medium. Commonly, the sample will contain protein or cell particles of very similar chemical characteristics, characteristics which would be impossible to separate one from another but for the electrophoresis separation technique. Under the influence of the electrical field within the apparatus, the electrophoretic mobilities of the various components of the sample will be different. Under the influence of the electrical field, the individual particles are rapidly accelerated in the lateral direction to approach a terminal velocity which is in equilibrium with the force of the viscous drag on the individual components. Each component will, in general, have a different lateral terminal velocity. A sample continuously introduced at some point into the sheet of liquid carrier medium (buffer) will flow in a narrow band in the absence of a potential gradient upon the apparatus. However, when a potential gradient is applied to the buffer sheet, the sample particles are separated under the influence of electrical field into the various components, depending upon the electrophoretic mobility of the respective components, the strength of the electrical field, and the length of time that the particles remain in the apparatus. Particles of similar mobility are concentrated in distinctive zones or bands which fan out from the point of sample introduction.
The present invention relates in particular to a free flow continuous electrophoresis process in which a buffer solution is made to flow freely in a uniform film or sheet through a central separation chamber defined by two parallel elongate plates. A sample is introduced into the buffer sheet at some point near the inlet to the separation chamber and an electrical potential gradient is applied across this flowing sheet perpendicular to the direction of buffer flow. The individual components within the sample then separate into narrow bands, depending upon their respective electrophoretic mobilities, and are collected from the outlet end of the separation chamber through one or more of a plurality of small tubes disposed along a collection manifold at the outlet of the separation chamber.
In present electrophoresis separation apparatus, the required electrical field is established between a pair of electrodes, one along a first lateral edge of the central separation chamber, and the other along the other lateral edge of the separation chamber. Because the buffer is usually made up of water with additional ionic species that, by their buffering action, protect the viability of the biological material being separated, the buffer fluid is electrically conductive. When the electrical field is imposed upon the conductive buffer carrier fluid, electrolysis of the water occurs, liberating hydrogen gas at the cathode electrode and oxygen gas at the anode electrode. Since the amount of gas liberated usually exceeds the amount that can be dissolved in the carrier buffer flow, undesirable byproducts are introduced into the buffer flow stream. This problem is circumvented in conventional electrophoresis separation apparatus by separating the lateral electrode chambers from the central separation chamber by ion permeable membranes and also by providing for locally increased buffer flow rates within the electrode chambers. Nevertheless, the utilization of ion permeable membranes between the electrode chambers and the central separation chamber creates several important limitations upon the separation process. Ionic species population in the buffer fluid will vary locally due to the polarization of the ion permeable membranes. The inherent electrical resistance of the membranes will create local heating of the buffer fluid. Also, pressure differentials will be created across the membranes due to electro-osmotic pumping within the separation chamber.